1) Field of the Invention
The present invention relates to a method for the preparation and purification of IgY(xcex94Fc) antibody from avian yolk, and the IgY(xcex94Fc) antibody produced thereby. The present invention also relates to uses of the novel IgY(xcex94Fc) antibody for quantitatively or qualitatively analyzing an etiological agent of interest.
2) Description of the Related Art
Antibodies are used widely in many biological investigations and clinical applications. Sera obtained from hyperimmunized mammalians are the most common source of polyclonal antibodies. Antibodies derived from such immune sera belong to a group of proteins called xe2x80x98immunoglobulins,xe2x80x99 among which the immunoglobulin G (IgG) is the most abundant. The IgG molecule consists of three domains, namely two Fab regions and one Fc region. The Fab portion involves mainly in antigen binding. The Fc portion, though having no ability to bind with an antigen, directs several biological activity of an antibody, such as complement fixing and Fc receptor binding.
In the art of immunodiagnostics, an intact IgG molecule is not suitable for use in detection systems and immunological assays involving mammalian sera since the Fc region on an IgG molecule is capable of binding to Fc receptors, activating the complement system, and reacting with rheumatoid factor in mammalian sera. Removal of the Fc portion of an IgG molecule frequently lead to a reduction in the interference (E. Lamoyi, Methods in Enzymology 121:652-663. (1986)).
Some of the suggested uses of antibody in immunotherapy include treating patients with intoxicated bacterial toxins or snake venoms (see, for example, U.S. Pat. Nos. 5,340,923 and 5,601,823), and protection of neonatal piglets against fatal enteric colibacillosis (see, for example, H. Brussow et al., J. Clin. Microbiol. 25:982 (1987); and C. O. Tacket et al., New Eng. J. Med. 318:1240 (1988)). Since the Fc fragment of an antibody molecule is known to be the most antigenic portion of the immunoglobulin (E. M. Akita et al., J. Immunol. Methods. 162:155-164 (1993)), cleavage of the same which results in the formation of an F(abxe2x80x2)2 fragment will reduce significantly a number of potential allergenic sites on the immunoglobulin molecule and is thus beneficial to human or an animal administered with the immunoglobulin.
Recently, the divalent F(abxe2x80x2)2 antibody fragment has been shown to be more useful in the immunodiagnostic tests (M. Muratsugu et al., J. Colloid Interface Sci 147:378 (1991); and J. L. Ortega-Vinuesa et al., J. Immunol Methods 90:29 (1996)) and more suitable for development of the immunoassays involving mammalian sera than the parent IgG.
The F(abxe2x80x2)2 antibody fragment, however, has not found widespread use in clinical immunodiagnostic kits as one might expect. This may be attributed to the difficulties and cost-ineffectiveness of large scale production of the F(abxe2x80x2)2 fragments, which is conventionally made by pepsin digestion of IgG and subsequent purification via chromatography.
Ducks and their phylogenetically close relatives and some reptiles, such as turtles, have three kinds of serum immunoglobulins: a macromolecular immunoglobulin IgM (800 kDa in duck), and two isoforms of low molecular weight IgG with sedimentation coefficients of 7.8S (in duck, 180 kDa) and 5.7S (in duck, 130 kDa), respectively. (E. R. Unanue etal., J. Exp. Med. 121:697-714 (1965); H. M. Grey, J. Immunol 98:811-819 (1967); and B. Zimmerman et al., Biochemistry 10:482-448 (1971)). Avian IgG is oftentimes called IgY due to their existence in egg yolk. The 5.7S IgY, constituted with shorter heavy chains, is structurally and antigenically similar to the F(abxe2x80x2)2 fragment of the 7.8S IgY (FIG. 1), and this fact leads to the nomenclature of IgY (equivalent to 7.8S IgY) and IgY(xcex94Fc) (equivalent to 5.7S IgY) to represent both isoforms of IgY (K. E. Magor et al., J. Immunol. 149:2627-2633 (1992)).
Studies conducted in the infected or experimentally immunized birds showed that duck antibodies are deficient in a number of biological effector functions, including complement fixation and Fc receptors binding, without sacrificing their binding activity to corresponding antigens (G. W. Litman et al., Immunochemistry 10:323 (1973); and T. E. Toth et al., Avian Dis. 25:17-28 (1981)). This may reasonably result from the apparent lack of Fc-equivalent region of the IgY(xcex94Fc) antibody that constitutes the quantitatively major component of duck antibody response. It is thus believed that the IgY(xcex94Fc) antibody, which appears to be a structural and functional analog of the F(abxe2x80x2)2 fragment, would provide magnificent advantages in immunological uses, if a promising process for manufacturing the antibody could be found, and the appropriate physical requirements for its activity could be identified.
Avian yolk antibodies have been reported to exhibit useful properties for both research and clinical applications as mammalian antibodies do (see, for example, U.S. Pat. Nos. 5,340,923; 5,585,098; 5,601,823; and 5,976,519). Egg yolks derived from a laying hen is inexpensive and more convenient and safer to handle as compared to the hyperimmunized mammalian sera. More importantly, yolk antibodies are able to stand up to the scrutiny under modern animal protection regulations (A. Polson et al., Immunol. Commun. 9:475 (1980); and B. Gottstein et al.). These facts suggest a potential use of egg yolk as a commercial source of antibodies.
Efforts have been made to isolate and purify IgY from egg yolks. For instance, materials, including agar, pectin (Japanese Kokai No. 64-38098 published in Feb. 8, 1989), dextran sulfate (J. C. Jensenius et al., J. Immunol. Methods 46:63 (1981)), natural gums (H. Hatta et al., J. Food Science 53:425(1988)) and polyethyl glycol (PEG) (A. Polson et al., Immunol. Invest. 14:323 (1985); see also U.S. Pat. No. 4,550,019 issued to A. Polson) were used to precipitate non-aqueous bio-molecules, mainly lipids and yolk granules, to thereby harvest a water soluble phase containing abundant yolk antibodies. A. Hassl et al. developed a two-step chromatographic process, comprised of hydrophobic interaction chromatography and size exclusive chromatography, for further isolation of yolk antibodies from a PEG-purified fraction (A. Hassl and H Aspock, J. Immunol. Methods 110:225 (1988)). Akita et al. described an improved method for isolating IgY, in which yolk antibodies were extracted from chick eggs by diluting the egg yolks with a large volume of water and subjecting the resultant supernatant to size exclusive chromatography and/or ion exchange chromatography (E. M. Akita et al., J. Immunol. Methods. 160:207 (1993); and E. M. Akita and S. Nakai, J. Food Sci. 57:629 (1993)).
However, all these studies and patents only focus on the isolation of the entire population of yolk antibodies (which includes at least IgY and IgY(xcex94Fc)) from avian eggs, rather than on the purification of IgY(xcex94Fc) antibody alone. Moreover, since the IgY(xcex94Fc) antibody is only present in the birds belonging to Order Anseriformes, including duck and goose, the isolation methods implemented on the galliform birds, such as chicken and turkey, provide no suggestion of a successful purification of IgY(xcex94Fc) antibody.
In 1989, Higgins tried to prepare the antibodies from hyperimmunized duck sera, but the antibodies affinity-purified at pH 8.0 and 0.5 M NaCl generally failed to exhibit effective precipitation or agglutination reactions (D. A. Higgins, Comp. Biochem. Physiol. 93B:135-144 (1989)). The optimal pH value for forming duck antibody precipitins, as Higgins alleged in the literature, ranges from pH 8.5 to pH 9.05. Since then, no critical study regarding the isolation of the IgY(xcex94Fc) antibody and its potential uses has been reported.
Therefore, there is a need in the art for a rapid, cost-effective, high-throughput process that provides easy isolation of the desired IgY(xcex94Fc) antibody from the antibody pool while maintaining its activity. Furthermore, there is a need in the art for a substantially purified IgY(xcex94Fc) which acts as a new type of F(abxe2x80x2)2 antibody for various immunodiagnostic and immunotherapeutic uses.
An extensive research has been conducted to fulfill the industrial requirements for yolk antibodies as indicated above. Surprisingly, we found that a successful isolation of IgY(xcex94Fc) antibodies from avian yolks can be readily accomplished through a simplified procedure under an optimal binding condition for the interaction between the antigen and antibody during immunoaffinity purification. By way of the process according to the present invention, a new type of F(abxe2x80x2)2 antibodies, i.e., the essentially purified IgY(xcex94Fc), can be easily manufactured with high yield in an economic manner, and the IgY(xcex94Fc) antibodies so produced are ready for a wide variety of immunological uses.
Accordingly, an object of the present invention is to provide a process for preparing IgYxcex94Fc antibodies from eggs laid by hens. The process generally comprises the steps of immunization of a fowl hen with an immunogen, a partial purification of the whole antibodies from the eggs laid by the hen, and an immunoaffinity purification of the antibodies raised against the immunogen, in which the binding of the antibodies with the immunogen/antigen in the immunoaffinity purification step is conducted in an environment of weak acid and low ionic strength. Specifically, the antibody-antigen interaction is conducted at pH within a range of 4-7 and under an ionic strength of lower than 50 mM, to obtain an optimal result.
The present invention thus provides a process for preparing IgYxcex94Fc) from egg yolk, which comprises the steps of:
(a) immunizing a fowl hen with a selected antigen so that fowl antibodies elicited against the antigen are accumulated in egg yolk;
(b) collecting egg yolk of the immunized hen and removing non-aqueous bio-molecules and granules therefrom to thereby obtain a water-soluble fraction containing egg yolk antibodies;
(c) passing the water-soluble fraction through an inert support matrix immobilized with the antigen thereon at pH within a range of 4-7 and under an ionic strength of lower than 50 mM to allow the formation of immuno-complexes of the immobilized antigen and the yolk antibodies; and
(d) dissociating and eluting out the yolk antibodies from the immobilized antigen.
Another object of this invention is to provide a novel IgY(xcex94Fc) antibody manufactured by the process according to this invention. It is still another object of the invention to provide the clinical and research uses of the IgY(xcex94Fc) antibody so produced. In addition to the cost-effectiveness and ease of preparation, the IgY(xcex94Fc) antibody according to the present invention has advantages of being inactive to the complement system and rheumatoid factors in mammalian sera, and having poor cross-reactivity to mammalian IgG, and is thus particularly suitable for use in immunological assays involving mammalian sera with minimal interference. As known by those skilled in the art, the antibody can be present in the form of a single reagent for clinical, research and other applications, or included in a commercial kit as an active component.
It is another specific object of the invention to provide a reagent for immunoassay of an etiological agent of interest, comprising an IgY(xcex94Fc) antibody manufactured by the process according to this invention.
A still another object of this invention is to provide a method of immunoassay and a commercial kit for conducting said immunoassay, in which an antibody, preferably an IgY(xcex94Fc) antibody purified according to the invention, against an etiological agent is incubated in the presence or absence of the etiological agent of interest under an optimal condition for binding to the etiological agent, to thereby quantitatively or qualitatively analyzing the etiological agent. According to the present invention, the optimal condition is at pH within a range of 4-7 and under an ionic strength of lower than 50 mM.